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C:\Documents and Settings\Sir\D Annals of Phytomedicine 4(1): 94-98, 2015 ANNALS OF PHYTOMEDICINE 94 Journal homepage: www.ukaazpublications.com An International Journal ISSN : 2393-9885 Evaluation of phytochemical investigation and immunomodulatory activity of four different plant species of vidari by carbon clearance test on wister rats V.K. Shilpashree, Raman Dang* and Kuntal Das* Karavali College of Pharmacy, NH-13, Opp: Mangalajyoti, Vamanjoor, Mangalore-575028, Karnataka, India *Department of Pharmacognosy and Phytochemistry, Krupanidhi College of Pharmacy, #12/1, Chikka Bellandur, Carmelaram Post, Varthur Hobli, Bengaluru-560035, Karnataka, India Received April 20, 2015: Revised May 10, 2015: Accepted May 15, 2015: Published online June 30, 2015 Abstract The present study was undertaken to evaluate immunomodulatory activity of vidari. Vidari is the name of four botanical sources of plant drug which is controversial in Indian market. They are namely; Pueraria tuberose DC (P.t) (Family: Leguminosae), Ipomoea mauritiana Jacq (I.m) (Family: Convolvulaceae), Adenia hondala de Wide (A.h) (Family: Passifloraceae) and Cycas circinalis Linn (C.c) (Family: Cycadaceae). In this study, preliminary investigations on phytoconstituents were evaluated separately and revealed the presence of carbohydrate, phenols, alkaloids, glycosides, saponins, phytosterol, flavonoids, wax and gums in all the examined drugs but tannins were absent in all the drugs. Furthermore, immune response was evaluated by carbon clearance assay (Granulopectic index) against standard marketed drug, Withania somnifera (W.s), an approved potent immunomodulatory agent (positive control). Wister rats of either sex were divided into 6 groups (6 animal each) and all the drugs were administered at dose of 250 mg/kg body wt. after calculated individual maximum tolerance dose as per OECD guidelines. All the test substances and standard showed significant activity compared to control (p < 0.001) and, thereafter, immune response with respect to granulopectic index of P.t (p > 0.05) was found better (0.051/ min), compared to other extracts but the value was lesser than that of positive control (0.053/ min). This study ascertained pharmacologically, the authenticity of four different species of plant belong to the same name of “vidari”. Key words: Carbon clearance assay, immunomodulatory, phytochemicals, vidari, Withania somnifera 1. Introduction of herbal medicines. Dried plant products sold in the market are generally difficult to identify and at the same time, identification of A vast number of research investigations have described the use of first step in quality control of herbal medicines. Different herbal plants in traditional ayurvedic medicine for many years and have raw drugs are sold under the same name and create controversies recently gained tremendous focus in the field of pharmacological with respect to the botanical identities of drugs, available in the industries. The quality assessments of the biological properties of market. Among the plants known for medicinal value, the ayurvedic extracts from various plants can serve as a source of newer drug plants with the name of vidari are very important for their molecules which help in many areas of health related problems therapeutic potentials. Vidari has four different plant species with (Acuna et al., 2009) and several marketed plant products have been their respective botanical identities and family (Venkatasubramanian used for the treatment of different diseases (Figueroa et al., 2015). et al., 2009), namely; Pueraria tuberose DC (P.t) (Family: Fabaceae), Many herbs enlisted as “Rasayana” drugs in Ayurveda are believed Ipomoea mauritiana Jacq (I.m) (Family: Convolvulaceae), Adenia to improve defense mechanisms of the body, promote physical and hondala de Wide (Family: Passifloraceae) and Cycas circinalis Linn mental health and enhance longevity (Shukla et al., 2009; Jantan et (C.c) (Family: Cycadaceae). The substitutes may or may not al., 2011; Kumar et al., 2012; Zhuang et al., 2012), among them resemble vidari in terms of morphology, properties or actions. Vidari Ocimum sanctum, W.s is well established as potent immune is an ingredient of Chyavanaprash, one of the top-selling products response plant drug (Caroline Jeba et al., 2011). In order to meet of ayurvedic industry (Venkatasubramanian et al., 2009). As per the rising demand for the raw drugs, adulteration and substitution the Ayurvedic Pharmacopoeia of India correlates vidari to P.t which have become frequent which in turn results in compromised quality is commonly known as “Vidarikand”. The plant is available throughout the India and it is traditionally used for bleeding disorders, decreased seminal quantity, purification of blood, tuberculosis, Author for correspondence: Dr. Kuntal Das Associate Professor, Krupanidhi College of Pharmacy, #12/1, Chikka cough, pain, burning micturition, herpes, rejuvenator, tonic, Bellandur, Carmelaram Post, Varthur Hobli, Bengaluru-560035, restorative, aphrodisiac, galactagogue, diuretic, demulcent, Karnataka, India haemorrhage, bronchial asthma and urinary disorders E-mail: [email protected] (Yoganarasimhan, 1996; The Ayurvedic Pharmacopoeia of India, Tel.: +91-9632542846 2006; Maji et al., 2014). But as per the local names of vidari, it Copyright @ 2015 Ukaaz Publications. All rights reserved. Email: [email protected]; Website: www.ukaazpublications.com 95 contains another three plants and they are used for different Mice (fasted for 3-4 h.) were divided into four groups, of two purposes like, tubers of I.m, A.h are used against sexual debility, animals (male and female) each for administration of each crude pain, inflammations, burning sensations and skin problems (Puri, drug. Mice were administered with test substances orally at the 2003) and C.c is used for high blood pressure, headaches, congestion, dose of 1g/kg body weight. Then the animals were observed for rheumatism, bone pain and treatment of estrogen-dependent tumors 24 h. for sign and symptoms of acute toxicity, viz. mortality, (Maria et al., 1995; Kalpashree and Raveesha, 2013). These activities tremors, alertness, eye movements and motility. Further, second are mainly due to the presence of several phytochemicals present dose of 2 g/kg body weight was given to the animals and observed in these plants, viz. puerarin, daidzein, genistein, genistein, for 72 h., which was also found to be safe and no toxic symptoms puerarone, coumarin, anthocyanin, lupinoside, tuberosin, were observed. W.s was used as standard at the dose of 250 mg/kg pterocarpintuberosin, puetuberosanol and hydroxytuberosone (Davis and Kuttan, 2000) and as per that recommended doses were (Maji et al., 2012; Sawale et al., 2013) among the phytochemicals, selected for all tested drugs at the dose of 250 mg/kg. the isoflavones puerarin, daidzein and genistein are most important for their immunomodulatory activity (Sawale et al., 2013) but no 2.6 Experimental protocol such concrete evidences are available for determination of the All the tested animals’, randomly divided into 6 groups of 6 rats pharmacological activity, especially comparative immunomo- each. dulatory activity for all four drugs. Based on the above context, the present study was designed to explore the immunomodulatory Group 1: Control animals potential of all four different plant drugs and to establish the potent Group 2: Pueraria tuberosa DC. (250 mg/kg) pharmacological authentication under the same local name of vidari. Group 3: Ipomoea mauritiana Jacq. (250 mg/kg) 2. Materials and Methods Group 4: Adenia hondala de Wide (250 mg/kg) 2.1 Plant material Group 5: Cycas circinalis L. (250 mg/kg) Group 6:Withania somnifera (L.) Dunal standard treated animals Dried mature tubers of P.t, I.m, A.h and stems of C.c used for the (250 mg/kg) study were collected by qualified field botanists from different locations of Belgaum, Bengaluru local market (Karnataka), 2.7 Carbon clearance assay Wayanad, Kozhikode, Idukki (Kerala), Pune, Bheamashanka All the animals in the 2nd, 3rd, 4th, 5th and 6th group received 250 mg/ (Maharashtra) and Tirupati (Andhra Pradesh). The samples were kg suspension of drugs, P.t, I.m, A.h, C.c and W.s for a period of 5 authenticated by qualified plant taxonomists of Herbarium division consecutive days. After 48 hours of last dose of drug, blood sample of FRLHT. Voucher Specimen No. (P.t = L/07/02/032; I.m = L/07/ 02/035; A.h = L/07/10/027 and C.c = L/07/04/011) for all the samples was collected by retro-orbital puncture which was taken as blank were preserved at Raw Drug Collection Centre, FRLHT, Bangalore. (Tiwari et al., 2004; Zhao et al., 2005). Each rat received an intravenous injection of carbon ink suspension consisting of 3 ml 2.2 Preparation of extract of pelikan ink (Germany), 4 ml of saline and 4 ml of 3% gelatin Dried coarsely powdered tubers of P.t, I.m, A.h and stem of C.c solution (Morris et al., 2003) via the tail vein at a dose of 0.5 ml/ (250 g) were separately defatted with petroleum ether at 45oC for 100 gm body weight (Daswani and Yegnanarayan, 2002). Blood 5 h, using Soxhlet apparatus. The marc left was subsequently was collected immediately after i.v injection of carbon ink extracted with ethyl acetate, methanol and water (45-50oC) for 7 h. suspension and at an interval of 3, 6, 9, 12, and 15 min. 25 µl of The crude brown residue mass of extract was then concentrated by blood was added to 2 ml of 0.1% sodium carbonate solution to lyse using rotary flash evaporator and then stored at 4oC in refrigeration the erythrocytes. At the end of the blood collection, the absorbance condition in separate glass bottles. The percentage yield of extracts of each sample was measured at 660 nm, using spectrophotometer. were calculated on dry wet basis. The clearance value ‘K’ was calculated according to the following 2.3 Pharmacognostic studies equation: K= (log OD ) – (log OD ) / t – t All the above extracted samples were screened for various 1 2 2 1 phytochemical constituents’ presence as per the method described where ‘t1’ represents the time in minutes when the samples ‘OD1’ by Kokate (1997).
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